This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Caspases (cysteine aspartyl proteases) are key enzymes responsible for the cellular remodeling of apoptosis, yet many of their cleavage targets remain unidentified. The Wells lab has undertaken proteome-scale in vitro studies of apoptotic remodeling process, and towards discovery of new cell death regulatory elements. While hundreds of caspase substrates have been identified in this manner, the functional relevance of each individual cleavage event remains unknown. It is likely that some cleavages are vital for apoptosis to proceed safely and efficiently, while others have little effect on the process of apoptosis and may in fact be "bystander" cleavages. We propose to distinguish between these two types of cleavages by evaluating their conservation across species. We will first study cells of Drosophila melanogaster contain caspases with high sequence similarity to human caspases, and undergo apoptosis that is morphologically similar to human cell apoptosis. We will also examine caspase cleavages in mouse cells, and in C. elegans whole worm extract. We will use the N-terminal labeling approach described by Mahrus et al. (2008) to enrich for protease substrates and identify them by mass spectrometry. With this approach, we are taking a key step towards addressing the question of the functional relevance of caspase cleavages on a large scale.